New York Blood Center Inc

COMPLICATIONS OF HEMOLYSIS AND TRANSFUSION THERAPY

PROGRAM PROJECT: RED CELL MEMBRANE STUDIES

DESIGN OF INHIBITORS TARGETED TO THE CD4 BINDING SITE ON HIV-1 GP120

THE DEVELOPMENT OF A RECOMBINANT VACCINE AGAINST HUMAN ONCHOCERCIASIS

DEVELOPMENT OF A NOVEL ADJUVANT FOR VACCINE SPARING

IMMUNE PATHOPHYSIOLOGY OF SICKLE CELL DISEASE - PROJECT ABSTRACT A CHRONIC INFLAMMATORY STATE IS NOW CONSIDERED A HALLMARK OF SICKLE CELL DISEASE (SCD) RESULTING FROM ONGOING HEMOLYTIC INSULT TO THE UNDERLYING VASCULATURE AND REPEATED CYCLES OF VASO-OCCLUSION CRISIS (VOC). TRANSFUSIONS REMAIN A CORNERSTONE TREATMENT FOR PATIENTS WITH SCD, BUT UNLIKE OTHER TRANSFUSED PATIENT POPULATIONS, A HIGHER PROPORTION (20-50%) OF PATIENTS WITH SCD DEVELOP ALLOANTIBODIES WITH POTENTIALLY LIFE-THREATENING SEQUALAE. THESE INCLUDE POORLY UNDERSTOOD DELAYED TRANSFUSION REACTIONS (DHTRS) WHOSE OCCURRENCE, AS WELL AS CLINICAL PROGRESSION FROM MILD TO SEVERE, IS UNPREDICTABLE. A LONG-STANDING INTEREST OF OUR RESEARCH PROGRAM HAS BEEN TO UNDERSTAND THE UNDERLING IMMUNE MECHANISMS LEADING TO SCD COMPLICATIONS INCLUDING VOC AND TRANSFUSION COMPLICATIONS. OUR STUDIES HAVE UNCOVERED AN UNDERAPPRECIATE ROLE OF CIRCULATING MONOCYTES AND THE INNATE IMMUNE RESPONSE TO HEMOLYSIS, A HALLMARK OF SCD, IN TRANSFUSION COMPLICATIONS INCLUDING ALLOIMMUNIZATION AND DHTRS. OUR PROJECTED STUDIES AS PART OF THIS R35 WILL BE FOCUSED ON INNATE IMMUNE CELLULAR PATHWAYS THAT MAGNIFY THE INFLAMMATORY SCD STATE VERSUS THE CELLS' HEME PROTECTIVE PATHWAYS SUCH AS THE HEME DETOXIFYING KEY ENZYME HEME OXYGENASE 1 IN ALLOIMMUNIZATION AND IN THE DEVELOPMENT OF DHTRS. OUR LABORATORY HAS ALSO IDENTIFIED A NOVEL FUNCTION FOR A SUBSET OF CIRCULATING MONOCYTES, REFERRED TO AS PATROLLING MONOCYTES (PMO), IN THEIR ABILITY TO SCAVENGE AND REMOVE DAMAGED ENDOTHELIUM AND ENDOTHELIAL-ATTACHED SICKLE RBCS. THE DISCOVERY OF A NOVEL MECHANISM OF ACTION OF PMO IN SCD OFFERS A PARADIGM SHIFT IN OUR UNDERSTANDING OF VOC AND OPENS UP THE POSSIBILITY THAT PMOS CAN BE MANIPULATED TO PREVENT PAINFUL VOC. AN OBJECTIVE OF THE PROPOSED STUDIES IN THIS R35 IS TO UNDERSTAND THE MECHANISMS BY WHICH PMO PROTECT SCD VASCULATURE AND HOW THEY ARE AFFECTED BOTH FUNCTIONALLY AND NUMERICALLY DURING A VASO-OCCLUSIVE EVENT WITH THE GOAL TO HELP ESTABLISH THEIR ROLE IN VOC PATHOPHYSIOLOGY, AND THEIR POTENTIAL AS A THERAPEUTIC TARGET FOR VOC. AS WE HAVE DONE IN THE PAST, ALL THE PROPOSED STUDIES FOR BOTH OBJECTIVES WILL COMBINE THE USE OF IN VIVO MOUSE MODELS, IN VITRO HUMAN MODEL SYSTEMS, AND PATIENT SAMPLES TO MECHANISTICALLY DISSECT IN A RIGOROUS MANNER HOW SPECIFIC IMMUNE CELL TYPES AND MOLECULAR PATHWAYS CONTRIBUTE TO SCD ALLOIMMUNIZATION AND VOC. ALTOGETHER, WE BELIEVE THAT OUR LABORATORY'S APPROACH OF INTEGRATING STUDIES IN HUMANS AND EXPERIMENTAL MODELS TO MECHANISTICALLY DISSECT IMMUNE PATHWAYS THAT REGULATE SCD COMPLICATIONS IS LIKELY TO BE IMPACTFUL AND GENERATE NOVEL FINDINGS IN AREAS CRITICAL TO THE NHLBI SCIENTIFIC MISSION.

RESTORING AGE-DEPENDENT VACCINE UNRESPONSIVENESS BY A NOVEL ASP-1 ADJUVANT COMBINATION - PROJECT ABSTRACT INFECTIOUS DISEASES ARE A MAJOR CAUSE OF MORBIDITY AND MORTALITY IN THE EXPANDING OLDER POPULATION. WHILE VACCINES ARE EFFICIENT MEASURES TO PREVENT INFECTIONS, A CRITICAL PROBLEM IS THAT AGING-ASSOCIATED CHANGES IN THE IMMUNE SYSTEM LEAD TO DECREASED IMMUNOGENICITY AND CLINICAL EFFICACY OF MOST CURRENTLY USED VACCINES. NOVEL STRATEGIES THAT INCREASE VACCINATION EFFICACY AND SPECIFICALLY TARGET THE AGED IMMUNE SYSTEM ARE IMPERATIVE. AN ESSENTIAL FACET OF THESE EFFORTS IS THE USE OF COMBINATORY ADJUVANTS THAT SYNERGISTICALLY POTENTIATE MORE EFFECTIVE VACCINE-INDUCED IMMUNE RESPONSES AGAINST VARIOUS PATHOGENS. IN THIS PROJECT WE WILL DEFINE THE MECHANISM OF ACTION (MOA) OF A COMBINATION OF A UNIQUE PARASITE PROTEIN ADJUVANT, RASP-1 WITH EACH OF THREE WELL- CHARACTERIZED INNATE ADJUVANTS: STING, TLR7/8 AND RIG-I AGONISTS, KNOWN TO ACTIVATE CYTOPLASMIC PATTERN RECOGNITION RECEPTORS AND TYPE I IFN SIGNALING, WHICH ARE SPECIFICALLY DAMPENED IN THE AGED POPULATION. WE HAVE SHOWN THAT RASP-1 IS A POWERFUL ADJUVANT AS IT ACTIVATES HUMAN AND MOUSE DENDRITIC CELLS TO POTENTIATE THE DIFFERENTIATION OF NAÏVE CD4+ T CELLS INTO TH1, TH17 AND TFH-LIKE CELLS. TRANSCRIPTOMIC ANALYSES OF RASP-1- ACTIVATED HMODCS REVEALED UPREGULATION OF THE MYD88-INDEPENDENT ACTIVATION PATHWAY AND INTERFERON RELATED GENES. IN MICE, RASP-1 ELICITS A BALANCED TH1/TH2 ANTIBODY RESPONSE, TH1-BIASED CELLULAR IMMUNITY, AND ENHANCED RESPONSES WHEN CO-ADMINISTERED WITH VACCINES LEADING TO INCREASED SURVIVAL FOLLOWING MICROBIAL CHALLENGE. IMPORTANTLY, OUR PRELIMINARY DATA INDICATE THAT RASP-1 IN COMBINATION WITH 2’3’-CGAMP (STING), R848 (TLR7/8) OR 5’3P-HPRNA (RIG-I) IN ACTIVATED ADULT AND AGED MOUSE BMDCS CAN DRAMATICALLY SYNERGIZE BEYOND MERELY ADDITIVE EFFECTS; IT ENHANCED SECRETION OF IL-12P40, IP-10 AND/OR IL-10. THE SYNERGY IS EXCEPTIONALLY EVIDENT WHEN IT ACTIVATES AGED MOUSE BMDCS WITH THE STING LIGAND. THEREFORE, WE HYPOTHESIZE THAT COMBINING RASP-1 WITH STING, TLR7/8 OR RIG-I AGONISTS WILL SYNERGISTICALLY ACTIVATE EARLY INNATE IMMUNE SIGNALING THAT CRITICALLY CONTRIBUTES TO THE ESTABLISHMENT AND NATURE OF IMMUNE RESPONSES, AND THE DURATION AND INTENSITY OF IMMUNE ACTIVATION. WE POSIT THAT THIS WILL HELP RESTORE THE AGING-ASSOCIATED DEFICITS IN THE CRITICAL REGULATORY PATHWAYS OF THE ANTIVIRAL RESPONSES. THROUGH COMPREHENSIVE IMMUNOLOGIC AND TRANSCRIPTOMIC ANALYSES, THE MOA THROUGH WHICH RASP-1 IN COMBINATION WITH THESE THREE PRR AGONISTS LEADS TO OPTIMAL ACTIVATION OF HELPER T CELLS DRIVING HUMORAL AND CELLULAR ANTIVIRAL RESPONSES WILL BE ESTABLISHED IN BOTH MOUSE (AIM 1) AND HUMAN (AIM 2) DCS (ADULT AND AGED), TO ESTABLISH THAT THEY ARE CONSERVED, AND THEN TEST THEIR EFFICACY IN VIVO IN ADULT AND AGED MICE USING THE TRIVALENT INACTIVATED INFLUENZA VACCINE (IIV3) AS A MODEL ANTIGEN (AIM 3). THIS PROJECT BRINGS TOGETHER A DYNAMIC TEAM WITH A HISTORY OF COLLABORATION AND COMPLEMENTARY EXPERTISE IN VACCINOLOGY, IMMUNOLOGY, BIOCHEMISTRY, INNATE CELL RECEPTOR BIOLOGY, ADJUVANTS, AND BIOINFORMATICS. WE WILL DELIVER A NOVEL, EFFICACIOUS COMBINATION OF INNATE CYTOPLASMIC PRR ADJUVANTS WITH RASP-1 THAT PROMOTES PROTECTIVE LONG-LIVED ADAPTIVE RESPONSES AND BOOSTS EFFICACY OF VACCINES IN AGED POPULATIONS.

LATEXIN FUNCTION IN THE MAINTENANCE AND REGENERATION OF THE HEMATOPOIETIC SYSTEM

RED CELL DEFORMABILITY INVITRO AND SURVIVAL INVIVO

NEIGHBORHOODS, NETWORKS, AND THE HIV CARE CONTINUUM AMONG HIV-INFECTED MSM IN NYC

ENHANCING POTENCY OF THE MERS VACCINE BY A NOVEL ASP-1+ALUM ADJUVANT COMBINATION

RATIONAL DESIGN OF ANTIVRIALS TARGETED TO HIV-1 CAPSID

USE OF FORWARD GENETIC APPROACHES TO IDENTIFY BABESIA RECEPTORS

T CELL IMMUNOREGULATION OF ALLOIMMUNIZATION IN SICKLE CELL DISEASE

HORMETIC ER STRESS REGULATION OF HEMATOPOIETIC STEM CELL FUNCTION - PROJECT SUMMARY HEMATOPOIESIS INVOLVES THE CONTINUOUS PRODUCTION OF RED BLOOD CELLS (ERYTHROCYTES), IMMUNE CELLS (LEUKOCYTES) AND BLOOD CLOTTING PLATELETS OVER THE LIFESPAN OF THE SUBJECT ALL OF WHICH ARE DERIVED FROM HEMATOPOIETIC STEM CELLS (HSCS) LOCATED IN BONE MARROW. HSCS HAVE TWO CRITICAL CHARACTERISTICS - MULTIPOTENCY AND SELF-RENEWAL. A COMPLETE PICTURE OF THE MOLECULAR MECHANISMS REGULATING HOMEOSTATIC OUTPUT OF BLOOD FROM HSCS IN VIVO HAS NOT YET EMERGED. OUR PUBLISHED FINDINGS SHOW HSCS ARE ENDOWED WITH LOW INTRACELLULAR CALCIUM (CA2+) COMPARED TO BONE MARROW (BM) PROGENITOR AND LINEAGE CELLS. WE HYPOTHESIZED A REDUCED EXTRACELLULAR CALCIUM ENVIRONMENT, SUCH AS A LOW CACL2 CULTURE MEDIA, MIGHT IMPROVE HSC FUNCTION. REMARKABLY, REDUCED CACL2 MEDIA DRAMATICALLY INCREASED PHENOTYPIC HSC COUNTS IN LONG-TERM CULTURES AND DEMONSTRATED A 20-FOLD INCREASE IN LONG-TERM DONOR ENGRAFTMENT COMPARED TO CLASSICAL CACL2 MEDIA FORMULATION, SUGGESTING FUNCTIONALLY POTENT HSCS ARE MAINTAINED IN LOW CACL2. WE DEMONSTRATED THAT HSC MAINTAIN LOW CA2+ LEVELS VIA HIGH EXPRESSION AND ACTIVITY OF PLASMA MEMBRANE CALCIUM EFFLUX PUMPS (PMCA) AND REDUCED BONE MARROW INTERSTITIAL FLUID CACL2 LEVELS. REDUCED CACL2 DECREASED MITOCHONDRIAL RESPIRATION, BUT NOT GLYCOLYSIS, SPECIFICALLY IN HSCS, WHILE INHIBITION OF GLYCOLYSIS ELEVATED HSC CA2+. THESE RESULTS DEMONSTRATE A POSITIVE FEEDBACK MECHANISM WHEREBY GLYCOLYTIC PMCA CA2+ EFFLUX ACTIVITY REDUCES CA2+ AND PREVENTS MITOCHONDRIAL RESPIRATION AND PROMOTES GLYCOLYSIS. CURIOUSLY, WE SHOWED MITOCHONDRIAL MASS IS HIGHEST IN HSCS SUGGESTING AN IMPORTANT, ALBIET RESPIRATION-INDEPENDENT, ROLE IN HSCS. BUILDING ON THESE FINDINGS, LITERATURE SUGGESTS REDUCED CACL2 CAN INDUCE ER STRESS. WE OBSERVED BCL-2 EXHIBITS A DOSE-DEPENDENT UPREGULATION UNDER REDUCED CACL2 CULTURE CONDITIONS IN HSCS, SUGGESTING INDUCTION OF A PRO-SURVIVAL GENE PROGRAM. REGULATORS OF THE ANTIOXIDANT GENES KNOWN TO BE INDUCED BY ER STRESS, INCLUDING ATF4 AND NRF2, MEDIATE UPREGULATION OF ANTIOXIDANT GENES INCLUDING BCL-2. WE FOUND ATF4 AND NRF2 WERE ALSO HIGH EXPRESSED IN HSCS IN LOW CACL2 CULTURE. THEREFORE, WE HYPOTHESIZE THAT REDUCED CACL2 INDUCES A HORMETIC ER STRESS RESPONSE THAT SUPPORTS HSC MAINTENANCE IN VITRO. WE PROPOSE TO STUDY WHAT TYPES OF ER STRESSES OCCUR AND WHICH UNFOLDED PROTEIN RESPONSE (UPR) SIGNALING PATH BRANCHES ARE ACTIVATED IN RESPONSE LOW CACL2. WE PROPOSE TO CHARACTERIZE THE TRANSCRIPTIONAL PROGRAM REGULATED BY THE PERK BRANCH OF UPR, WHICH ACTIVATES THE ATF4/NRF2 ER STRESS RESPONSE PROGRAMS, THAT WE HAVE IDENTIFIED TO BE ACTIVE. FURTHER, WE PROPOSE TO STUDY THE UPREGULATION OF BCL-2 IN HSCS UNDER LOW CACL2 CONDITIONS TO DETERMINE IF A NON-CANONICAL ROLE FOR BCL-2 INHIBITS IP3R RELEASE OF CA2+ FROM LUMINAL ER CA2+ STORES IN HSCS. FURTHERMORE, WE PROPOSE TO CORROBORATE THESE FINDINGS IN HUMAN CB HSCS TO ACCELERATE TRANSLATIONAL POTENTIAL OF THE FINDINGS. THIS WOULD ESTABLISH A NOVEL POSITIVE FEEDBACK LOOP REQUIRED TO REDUCE CA2+ LEVELS AND PRESERVE SELF-RENEWAL AND MULTIPOTENCY IN HSCS. THESE FINDINGS WILL EXPAND OUR FUNDAMENTAL UNDERSTANDING OF HSC BIOLOGY AND MAY INSPIRE METHODS TO IMPROVE HSC EXPANSION FOR CLINICAL APPLICATIONS.

TRANSFUSION-DRIVEN HYPERHEMOLYSIS IN SICKLE CELL DISEASE - RED BLOOD CELL (RBC) TRANSFUSIONS REMAIN A CORNERSTONE TREATMENT IN THE MANAGEMENT OF SICKLE CELL DISEASE (SCD). HOWEVER, PATIENTS MAY EXPERIENCE DELAYED HEMOLYTIC TRANSFUSION REACTION (DHTR) WHICH IN THIS PATIENT POPULATION HAS AN UNPREDICTABLE PROGRESSION FROM MILD TO LIFE-THREATENING SEVERE REACTIONS WHERE BOTH TRANSFUSED AND PATIENT’S OWN RBCS ARE DESTROYED ALONG WITH RETICULOCYTOPENIA AT THE TIME OF HEMOLYTIC CRISIS, EXACERBATING THE ANEMIA. THE MECHANISMS UNDERLYING SEVERE DHTR PROGRESSION ARE POORLY UNDERSTOOD, POSING CHALLENGES FOR PREVENTION AND EFFECTIVE TREATMENTS FOR THIS TRANSFUSION COMPLICATION WHICH DISPROPORTIONATELY IS ENCOUNTERED IN PATIENTS WITH SCD. WE RECENTLY FOUND THAT ACUTE HEMOLYSIS INDUCES TYPE I INTERFERON (IFN-I) PRODUCTION IN INNATE IMMUNE CELLS, LEADING TO INCREASED DIFFERENTIATION AND ACTIVATION OF MONOCYTE-DERIVED MACROPHAGES (MOMF) IN SCD, AND EXACERBATING DESTRUCTION OF ANTIBODY (AB)-COATED TRANSFUSED RBCS. OUR PRELIMINARY DATA SHOWED THAT AB-SENSITIZED RBC DESTRUCTION ALONE ALSO LED TO IFN-I PRODUCTION. INTERESTINGLY, ONLY WHEN THE INCOMPATIBLE RBC DESTRUCTION OCCURS UNDER HEMOLYTIC CONDITIONS INDUCING HIGHEST LEVELS OF IFN-I, WE DETECT BYSTANDER HEMOLYSIS OF SICKLE RBCS, MIMICKING HYPERHEMOLYSIS REACTION IN SCD. WE ALSO FOUND THAT HEMOLYSIS-INDUCED IFN-I IMPAIRS ERYTHROPOIESIS ALONG WITH INHIBITION OF EPO/EPOR SIGNALING. BASED ON THESE DATA, WE HYPOTHESIZE THAT FC RECEPTOR CROSSLINKING IN A HEMOLYTIC BACKDROP OF SCD LEADS TO INCREASE IN IFN-I LEVELS, CAUSING HEIGHTENED IFN- I SIGNALING IN PHAGOCYTES AND ERYTHROID CELLS WHICH TRIGGER INCREASED RBC DESTRUCTION AND INHIBITS RBC PRODUCTION, RESPECTIVELY, IN SEVERE DHTR. IN AIM 1, WE WILL FOCUS ON IDENTIFYING MECHANISMS OF BYSTANDER HEMOLYSIS BY EXAMINING THE ROLE OF KEY PHAGOCYTOSIS ACTIVATION MOLECULES, INCLUDING THROMBOSPONDIN (TSP-1) AND ITS LIGANDS WHICH ARE UPREGULATED IN OUR HYPERHEMOLYTIC MODELS. WE WILL COMPARE THE ROLE OF AB-MEDIATED ERYTHROPHAGOCYTOSIS VERSUS AB-INDEPENDENT RBC ENGULFMENT IN TRIGGERING BYSTANDER HEMOLYSIS AND INTERROGATE THE RELATIVE CONTRIBUTION OF MURINE/HUMAN FCR/SYK PATHWAY AND HEME ACTIVATION PATHWAYS IN BYSTANDER HEMOLYSIS. WE WILL ALSO EXAMINE THE POTENTIAL OF IFN-I AS A BIOMARKER OF DHTR SEVERITY BY EXAMINING IFN- I/STAT1 DRIVEN CHANGES IN MONOCYTE PHAGOCYTOSIS ASSOCIATED PROFILES IN SCD PATIENT SAMPLES, COMPARING PATIENTS EXPERIENCING SEVERE AND MILD DHTR AND AFTER RECOVERY TO STEADY STATE. FOR AIM 2, WE WILL DEFINE THE MECHANISMS BY WHICH IFN-I SUPPRESSES ERYTHROPOIESIS USING PRIMARY ERYTHROID CELL CULTURE SYSTEM AND TARGETED DELETION OF KEY DOWNSTREAM PATHWAYS IN HUMAN ERYTHROBLAST CELL LINES AND MOUSE MODELS. WE WILL ALSO TEST THE THERAPEUTIC EFFECTS OF INHIBITING IFN-I PRODUCTION/IFN-I SIGNALING OR/AND INCREASING EPO/EPOR SIGNALING ON REVERSING IMPAIRED BM ERYTHROPOIESIS IN SCD MICE AND ON HUMAN ERYTHROPOIESIS IN VITRO AND IN CULTURES TREATED FROM SCD PATIENT PLASMA. WE BELIEVE THAT OUR PROPOSED STUDIES TO EXAMINE THE BASIS FOR PROGRESSION TO DHTR SEVERITY MAY HELP STRATIFY RISK AND AID IN DEVELOPMENT OF NOVEL TARGETED THERAPIES TO REVERSE OR PREVENT HYPERHEMOLYSIS, A DEVASTATING COMPLICATION OF AN OTHERWISE LIFE-SAVING TREATMENT IN SCD.

COMMUNITY FACTORS, HIV AND RELATED HEALTH OUTCOMES IN MEN WHO HAVE SEX WITH MEN

MECHANISMS CONTROLLING TRANSFUSION-ASSOCIATED ANTIBODY RESPONSES IN SCD ALLOIMMUNIZATION

BABESIOSIS: AN EMERGING INFECTIOUS THREAT TO TRANSFUSION MEDICINE

TESTING AN INTERVENTION TO INCREASE HIV SELF-TESTING AMONG YOUNG, BLACK MSM

IMMUNOPATHOLOGY OF ITP

USING TECHNOLOGY TO MATCH YOUNG BLACK MSM TO HIV TESTING OPTIONS

ERYTHROBLASTIC ISLAND MACROPHAGES IN NORMAL ERYTHROPOIESIS AND DISORDERED ERYTHROPOIESIS IN POLYCYTHEMIA VERA - ABSTRACT ERYTHROPOIESIS OCCURS AT THE ERYTHROBLASTIC ISLAND WHERE THE CENTRAL MACROPHAGE (EBI M) SUPPORTS PROLIFERATION AND MATURATION OF THE SURROUNDING ERYTHROID CELLS. ALTERATIONS IN EBI M CONTRIBUTE TO THE DISORDERED ERYTHROPOIESIS IN VARIOUS HEMATOLOGICAL DISEASES INCLUDING POLYCYTHEMIA VERA (PV) WHICH IS CHARACTERIZED BY ERYTHROCYTOSIS DUE TO V617F MUTATION IN JAK2 (JAK2V/F). IN THIS APPLICATION, WE PROPOSE TO DEFINE THE MECHANISMS BY WHICH EBI M SUPPORT NORMAL ERYTHROPOIESIS IN AIM 1 AND TO DEFINE THE MECHANISMS BY WHICH JAK2V/F IN EBI M CONTRIBUTES TO PATHOGENESIS OF PV IN AIM 2. OUR OVERALL HYPOTHESIS IS THAT EBI M SUPPORT NORMAL ERYTHROPOIESIS VIA EPOR-STAT5 SIGNALING-MEDIATED PRODUCTION OF GROWTH FACTORS IGF1 AND SCF AS WELL AS IRON CARRIER TRANSFERRIN AND THAT JAK2V/F-INDUCED CONSTITUTIVE ACTIVATION OF EPOR SIGNALING IN THE EBI M LEADS TO UPREGULATION OF TRANSFERRIN RECEPTOR CD71 WHICH CONTRIBUTES TO THE HYPERPROLIFERATION OF EBI M ALONG WITH INCREASED PRODUCTION OF IGF1 AND SCF LEADING TO OVERPRODUCTION OF RED BLOOD CELLS THAT CAN BE ALLEVIATED BY INTERFERING WITH CD71, IGF1-IGF1R AXIS OR/AND SCF-CKIT AXIS. OUR HYPOTHESIS IS BASED ON OUR FINDINGS THAT I) EBI M EXPRESSED EPOR; II) EPOR+ M EXPRESSED IGF1 AND SCF AS WELL AS IRON CARRIER TRANSFERRIN (TRF); III) EPO STIMULATION INCREASED THE LEVELS OF IGF1, SCF AND TRF BY EBI M; IV) SELECTIVE DELETION OF EPOR IN M LED TO ANEMIA ALONG WITH DECREASED LEVELS OF IGF1, SCF AND TRF; V) SELECTIVE DELETION OF IGF1 OR TRF IN M ALSO LED TO ANEMIA; VI) ALTHOUGH KNOCK-IN OF JAK2V/F IN ERYTHROID CELL ALONE USING GYPA-TDTOMATO-CRE OR IN M ALONE USING CD169- TDTOMATO-CRE LED TO ERYTHROCYTOSIS IN MICE, KNOCK-IN OF JAK2V/F IN BOTH ERYTHROID CELLS AND M WERE REQUIRED TO FULLY RECAPITULATE CHARACTERISTICS OF HUMAN PV; VII) KNOCK-IN OF JAK2V/F IN M LED TO INCREASED NUMBERS OF BM EBI M AND SPLEEN RED PULP M ALONG WITH INCREASED LEVELS OF TRANSFERRIN RECEPTOR TRFC (CD71), IGF1 AND SCF. IN AIM 1, WE WILL GENERATE CONDITIONAL KNOCKOUT MICE IS WHICH EPOR, STAT5, IGF1, KITL OR TRF IS SELECTIVELY DELETED IN M AND EXAMINE THEIR EFFECTS ON RED CELL PARAMETERS, FORMATION OF EBIS, CELLULAR AND MOLECULAR CHANGES OF BOTH EBI M AND ERYTHROID CELLS BOTH IN VIVO AND IN VITRO. WE WILL ALSO EXAMINE THE EFFECTS OF KNOCKDOWN OF THESE GENES IN HUMAN EPOR+ M ON HUMAN ERYTHROPOIESIS USING IN VITRO CO-CULTURE SYSTEM. IN AIM 2, WE WILL EXAMINE THE EFFECTS OF JAK2 INHIBITOR ON OUR PV MOUSE MODELS IN WHICH JAK2V/F WAS EXPRESSED IN . WE WILL EXAMINE WHETHER SELECTIVE DELETION OF TRFC, IGF1 OR/AND KITL IN  IN MICE CAN ALLEVIATE ERYTHROCYTOSIS OF PV MICE. WE WILL ALSO EXAMINE WHETHER SELECTIVE DELETION OF IGF1R AND CKIT (THE RECEPTOR FOR SCF) IN ERYTHROID CELLS CAN ALLEVIATE ERYTHROCYTOSIS. WE WILL USE CHEMICAL INHIBITORS TO INTERFERE WITH IGF1-IGF1R AXIS OR/AND SCF-CKIT AXIS TO EXAMINE THEIR EFFECTS ON ERYTHROCYTOSIS. WE WILL EXAMINE WHETHER THE CHANGES WE OBSERVED IN MOUSE EBI  ARE PRESENT IN HUMAN BM EBI  FROM PV PATIENTS. FINDINGS FROM THE PROPOSED STUDIES WILL PROVIDE NOVEL INSIGHTS INTO BASIC BIOLOGY OF ERYTHROPOIESIS, HAVE IMPLICATIONS IN UNDERSTANDING THE MECHANISMS OF ANEMIA AND ERYTHROCYTOSIS, HELP IMPROVING PROTOCOLS FOR EX VIVO RED CELL PRODUCTION AND REVEAL NOVEL THERAPEUTIC TARGETS FOR THE TREATMENT OF PV.

IMMUNOREGULATORY RESPONSES IN PATIENTS WITH ITP

RATIONAL DESIGN OF HIV FUSION INHIBITORS TARGETING GP41

PATROLLING MONOCYTES IN SICKLE PAIN CRISIS AND FOLLOWING TRANSFUSION

MECHANISM OF THE SHORT- AND LONG-TERM EFFECTS OF COVID-19-INDUCED ALARMINS ON HEMATOPOIETIC STEM AND PROGENITOR CELLS. - ABSTRACT THE LONG-TERM GOAL OF THIS PROPOSAL IS TO UNDERSTAND THE LONG-TERM SEQUALAE OF ACUTE COVID-19 INFECTION ON HEMATOPOIETIC AND IMMUNE DAMAGES, AND TO IDENTIFY THE KEY PATHWAYS AND MECHANISM BY WHICH COVID-19- ASSOCIATED CYTOKINE DYSREGULATION ALTERS HSC FUNCTION AND DIFFERENTIATION. SARS-COV-2 INFECTION CAUSES LOCAL AND SYSTEMIC DAMAGES DUE TO DYSREGULATED IMMUNE RESPONSE AND CYTOKINE PRODUCTION. ITS LONG-TERM NEGATIVE EFFECTS ON BODY TISSUE AND ORGAN REMAIN LARGELY UNKNOWN. OUR PUBLISHED WORK SHOWED THAT SARS-COV-2 INFECTION DRAMATICALLY INCREASED NEUTROPHIL PRODUCTION AND NEUTROPHIL-ASSOCIATED S100A8/A9 (ALARMIN) RELEASE. PERSISTENT HIGH LEVEL OF S100A8/A9 IS A NEGATIVE PROGNOSTIC BIOMARKER FOR THE DISEASE SEVERITY AND MORTALITY. ALTHOUGH THE FUNCTION OF S100A8/A9 ON MATURE BLOOD CELLS HAVE BEEN STUDIED, ITS FUNCTIONAL EFFECT ON HEMATOPOIETIC STEM CELLS (HSCS) ARE UNKNOWN. OUR PRELIMINARY DATA SHOW THAT S100A8/A9 CAUSES LOSS OF QUIESCENCE AND DIFFERENTIATION OF HSC TOWARD MYELOID PROGENITORS AT THE EXPENSE OF HSCS. TOLL-LIKE RECEPTOR 4 (TLR4), THE ENDOGENOUS RECEPTOR OF S100A8/A9, IS HIGHLY EXPRESSED IN HSCS, AND S100A8/A9 ACTIVATES ITS CANONICAL DOWNSTREAM MAPK (MITOGEN-ACTIVATED PROTEIN KINASE) PATHWAY. VERY INTERESTINGLY, S100A8/A9 CAUSES DOWNREGULATION OF EPIGENETIC REGULATOR SETD2, LEADING TO THE C-MYC UPREGULATION IN HSCS. C-MYC IS A KEY DOWNSTREAM TARGET OF BOTH MAPK AND SETD2 PATHWAYS. MAPK, SETD2 AND C-MYC ARE IMPORTANT REGULATORS OF HSC PROLIFERATION AND MYELOID DIFFERENTIATION. WE HYPOTHESIZE THAT SARS-COV-2-INDUCED S100A8/A9 ACTIVATES TLR4 SIGNALING WHICH CONVERTS TO C-MYC IN HSCS, RESULTING IN LOSS OF QUIESCENCE AND SELF-RENEWAL, MYELOID DIFFERENTIATION SKEWING, AND LONG-TERM IMPAIRMENT OF HEMATOPOIESIS. SINCE HSC IS RESPONSIBLE FOR THE LIFE-LONG PRODUCTION OF BLOOD CELLS, INCLUDING ALL TYPES OF IMMUNE CELLS, ANY FUNCTIONAL DAMAGES OF HSCS WOULD LATER ON HAVE PROFOUNDLY NEGATIVE EFFECTS ON THE IMMUNE RESPONSE. THEREFORE, UNDERSTANDING THE CELLULAR AND MOLECULAR MECHANISM BY WHICH S100A8/A9 REGULATES HSCS AND HEMATOPOIESIS WOULD CONTRIBUTE A NEW EVIDENCE BASE TO ACCELERATE ADVANCES IN DIAGNOSTICS, THERAPEUTICS, CLINICAL MANAGEMENT OF COVID-19 PATIENTS IN ACUTE INFECTION AND RECOVERY PHASES. 1

GENOMIC APPROACHES TO IMPROVE TRANSFUSION THERAPY IN SCD PATIENTS

SARS-COV S PROTEIN RECEPTOR-BINDING DOMAIN-BASED VACCINES

RHEOLOGICAL AND ADHERENCE PROPERTIES OF SICKLE CELLS

TET3 IN TERMINAL ERYTHROID DIFFERENTIATION

SEXUAL NETWORKS DRUGS AND HIV RISK AMONG MEN WHO HAVE SEX WITH MEN

DESIGNING A ROBUST PLATFORM FOR THE IN VITRO PROPAGATION OF BABESIA MICROTI IN HUMAN RBCS - THE OVERALL GOAL OF THE R61 PHASE OF OUR PROPOSAL IS TO ESTABLISH A CONTINUOUS IN VITRO CULTURE SYSTEM OF B. MICROTI IN HUMAN RBCS TO ENABLE A PLATFORM AMENABLE FOR FUTURE EXPERIMENTAL INVESTIGATION, SPECIFICALLY THE IDENTIFICATION AND CHARACTERIZATION OF HUMAN RBC RECEPTOR-B. MICROTI LIGAND INTERACTIONS THAT FACILITATE PARASITE INVASION WHICH WILL BE EXPLORED IN THE R33 PHASE. WE HYPOTHESIZE THAT B. MICROTI LIKE OTHER ERYTHROCYTE SEEKING PROTOZOANS HAVE PREFERENTIAL CELLULAR INVASION AND PROLIFERATION NEEDS. TO ESTABLISH IF THIS IS TRUE, WE WILL IN THE R61 PHASE, MILESTONE1, ANALYZE THE PARASITE-HOSTING CAPABILITIES OF SUB-POPULATIONS OF RBCS USING SEPARATION AND PURIFICATION PROTOCOLS FROM PERIPHERAL BLOOD, UMBILICAL CORD BLOOD AND HEMOCHROMATOSIS BLOOD DRAWS, USING PARASITE INOCULUM FROM BOTH B. MICROTI INFECTED MICE AND INFECTED HUMAN SOURCES. WE WILL ALSO ANALYZE PURE POPULATIONS FROM ERYTHROID PROGENITOR CELLS THAT HAVE BEEN FACS SORTED FOR THE ABILITY TO BE INFECTED WITH B. MICROTI. ONCE A SPECIFIC HOST RBC HAS BEEN IDENTIFIED ALONG WITH THE INOCULUM, WE WILL ASSAY CULTURE MEDIA ALONG WITH SERUM/LIPID ADDITIVES AND VITAMIN/MINERAL ADDITIVES, AND IDEAL GAS CONDITIONS, TO ACHIEVE MILESTONE 2. WORKING WITH THE HIGHEST INFECTION ACHIEVING MEDIA, HOST RBC AND PARASITE SOURCE, WE WILL MONITOR PARASITE EGRESS AND RE-INVASION AFTER ENSURING ADEQUATE HOST RBCS THAT THE PARASITE REQUIRES, IN CULTURE, IN MILESTONE 3. FACS AND GIEMSA ESTIMATION OF PARASITEMIA AND OVERALL CULTURE DYNAMICS WILL BE CONSTANTLY MONITORED, PAYING PARTICULAR ATTENTION TO SUB-POPULATION STRUCTURE OF THE CULTURES, WHICH WILL INFORM US OF SUCCESS OF VARIOUS MILESTONES. AS PART OF MILESTONE 4, WE WILL CRYOPRESERVE AND REVIVE STOCKS OF CULTURE-ADAPTED PARASITES. COMPREHENSIVE BIO-PROTOCOLS WILL BE WRITTEN UP DETAILING SPECIFIC ASPECTS OF EACH STEP IN THE CULTURING PROCESS. ONCE AN IN VITRO CULTURE SYSTEM IS IN PLACE CRITICAL ASPECTS OF HUMAN HOST-PARASITE RELATIONSHIPS CAN BE EXPLORED AND WE WILL INVESTIGATE B. MICROTI INVASION OF THE HUMAN RBC, IN THE R33 PHASE. TO IDENTIFY AND CHARACTERIZE RBC SURFACE MOLECULES THAT FUNCTION IN INVASION, WE WILL USE ENZYMATICALLY TREATED RBCS, CLINICALLY DEFINED RBCS DEFICIENT IN SPECIFIC RECEPTOR, INHIBITION OF INVASION ASSAYS USING ANTIBODIES/PEPTIDES AND FORWARD GENETIC APPROACHES USING SHRNA MEDIATED KNOCKDOWN OF SPECIFIC RBC RECEPTORS. COGNATE B. MICROTI LIGANDS WILL THEN BE IDENTIFIED USING GEL OVERLAY AND PULL-DOWN ASSAYS. VALIDATION OF THESE RECEPTOR-LIGAND INTERACTIONS WILL BE CARRIED OUT VIA AVEXIS AND BIACORE ASSAYS. FUNCTIONAL VALIDATION ASSAYS WILL INCLUDE INHIBITION OF INVASION ASSAYS WITH ANTI-LIGAND ANTIBODIES AND RECOMBINANT LIGAND PROTEINS. WE BELIEVE THAT THESE INNOVATIVE APPROACHES WILL GREATLY ENHANCE THE OPPORTUNITIES AND CAPABILITIES FOR INVESTIGATIONS INTO B. MICROTI BIOLOGY.

INFLUENCE OF NEIGHBORHOODS AND NETWORKS ON THE HIV CARE CONTINUUM AMONG HIV-INFECTED MSM IN NYC

ORGANELLE MEMBRANES IN PLATELET STORAGE POOL DISEASE

TRANSFUSION-DRIVEN HYPERHEMOLYSIS IN SICKLE CELL DISEASE - RED BLOOD CELL (RBC) TRANSFUSIONS REMAIN A CORNERSTONE TREATMENT IN THE MANAGEMENT OF SICKLE CELL DISEASE (SCD). HOWEVER, PATIENTS MAY EXPERIENCE DELAYED HEMOLYTIC TRANSFUSION REACTION (DHTR) WHICH IN THIS PATIENT POPULATION HAS AN UNPREDICTABLE PROGRESSION FROM MILD TO LIFE-THREATENING SEVERE REACTIONS WHERE BOTH TRANSFUSED AND PATIENT’S OWN RBCS ARE DESTROYED ALONG WITH RETICULOCYTOPENIA AT THE TIME OF HEMOLYTIC CRISIS, EXACERBATING THE ANEMIA. THE MECHANISMS UNDERLYING SEVERE DHTR PROGRESSION ARE POORLY UNDERSTOOD, POSING CHALLENGES FOR PREVENTION AND EFFECTIVE TREATMENTS FOR THIS TRANSFUSION COMPLICATION WHICH IS DISPROPORTIONATELY ENCOUNTERED IN PATIENTS WITH SCD. WE RECENTLY FOUND THAT ACUTE HEMOLYSIS INDUCES TYPE I INTERFERON (IFN-I) PRODUCTION IN INNATE IMMUNE CELLS, LEADING TO INCREASED DIFFERENTIATION AND ACTIVATION OF MONOCYTE-DERIVED MACROPHAGES (MOMF) IN SCD, AND EXACERBATING DESTRUCTION OF ANTIBODY (AB)-COATED TRANSFUSED RBCS. OUR PRELIMINARY DATA SHOWED THAT AB-SENSITIZED RBC DESTRUCTION ALONE ALSO LED TO IFN-I PRODUCTION BUT WITH EVEN HIGHER LEVELS IF DESTRUCTION OCCURRED UNDER HEMOLYTIC CONDITIONS, WHICH INTERESTINGLY ALSO INDUCED BYSTANDER HEMOLYSIS OF SICKLE RBCS, MIMICKING HYPERHEMOLYSIS REACTION IN SCD. WE ALSO FOUND THAT HEMOLYSIS-INDUCED IFN-I IMPAIRS ERYTHROPOIESIS ALONG WITH INHIBITION OF EPO/EPOR SIGNALING. BASED ON THESE DATA, WE HYPOTHESIZE THAT FC RECEPTOR CROSSLINKING IN A HEMOLYTIC BACKDROP OF SCD LEADS TO INCREASE IN IFN-I LEVELS, CAUSING HEIGHTENED IFN-I SIGNALING IN PHAGOCYTES AND ERYTHROID CELLS WHICH TRIGGER INCREASED RBC DESTRUCTION AND FURTHER SUPPRESSION OF RBC PRODUCTION, RESPECTIVELY, LEADING TO SEVERE DHTR. IN AIM 1, WE WILL FOCUS ON IDENTIFYING MECHANISMS OF BYSTANDER HEMOLYSIS BY EXAMINING THE ROLE OF KEY PHAGOCYTOSIS ACTIVATION MOLECULES, SPECIFICALLY SCD ASSOCIATED EAT ME SIGNALS INCLUDING THROMBOSPONDIN (TSP-1) AND ITS LIGANDS WHICH ARE UPREGULATED IN HYPERHEMOLYTIC MODELS. WE WILL COMPARE THE ROLE OF AB-MEDIATED ERYTHROPHAGOCYTOSIS VERSUS AB-INDEPENDENT RBC ENGULFMENT IN TRIGGERING BYSTANDER HEMOLYSIS AND INTERROGATE THE RELATIVE CONTRIBUTION OF INHIBITING FCR/SYK PHOSPHORYLATION AND HEME ACTIVATION PATHWAYS IN AUTOLOGOUS SICKLE RBC DESTRUCTION. WE WILL ALSO EXAMINE THE POTENTIAL OF IFN-I AS A BIOMARKER OF DHTR SEVERITY BY EXAMINING IFN-I/STAT1 DRIVEN CHANGES IN MONOCYTES IN SCD PATIENT SAMPLES, COMPARING PATIENTS EXPERIENCING SEVERE AND MILD DHTR AND AFTER RECOVERY TO STEADY STATE. FOR AIM 2, WE WILL DEFINE THE MECHANISMS BY WHICH IFN-I SUPPRESSES ERYTHROPOIESIS USING PRIMARY ERYTHROID CELL CULTURE SYSTEM AND TARGETED DELETION OF KEY DOWNSTREAM PATHWAYS IN HUMAN ERYTHROBLAST CELL LINES AND MOUSE MODELS. WE WILL ALSO TEST THE THERAPEUTIC EFFECTS OF INHIBITING IFN-I PRODUCTION/IFN-I SIGNALING OR/AND INCREASING EPO/EPOR SIGNALING ON REVERSING IMPAIRED BM ERYTHROPOIESIS IN SCD MICE AND ON HUMAN ERYTHROPOIESIS IN VITRO AND IN CULTURES TREATED WITH SCD PATIENT PLASMA. WE BELIEVE THAT OUR PROPOSED STUDIES TO EXAMINE THE BASIS FOR PROGRESSION TO DHTR SEVERITY MAY HELP STRATIFY RISK AND AID IN DEVELOPMENT OF NOVEL TARGETED THERAPIES TO REVERSE OR PREVENT HYPERHEMOLYSIS, A DEVASTATING COMPLICATION OF AN OTHERWISE LIFE-SAVING TREATMENT IN SCD.

DESIGN OF INHIBITORS TARGETED TO THE CD4 BINDING SITE ON HIV - 1GP120 - PROJECT SUMMARY/ABSTRACT THE HIV-1 ENVELOPE GLYCOPROTEIN (ENV) GP120 IS CRITICAL IN MEDIATING VIRAL ENTRY INTO HOST CELLS AND IS A PRIME TARGET FOR SMALL-MOLECULE DRUG AND VACCINE DEVELOPMENT. THE RECENT FDA APPROVAL OF THE ENV GP120–TARGETING FOSTEMSAVIR (RUKOBIA, VIIV HEALTHCARE) VALIDATES THIS PROTEIN AS A TARGET FOR DRUG DEVELOPMENT. HOWEVER, A 48- WEEK PHASE 2B CLINICAL TRIAL REPORTED SEVERAL RESISTANT MUTANTS THAT REDUCED SUSCEPTIBILITY TO FOSTEMSAVIR. THEREFORE, A CRITICAL NEED EXISTS FOR THE CONTINUED DEVELOPMENT OF NOVEL DRUGS AGAINST THIS TARGET FOR EFFECTIVE THERAPIES. OUR GROUP HAS MADE SIGNIFICANT ADVANCES TOWARD MEETING THIS URGENT NEED BY DEVELOPING A NEW CLASS OF HIV-1 ENTRY INHIBITORS TARGETING THE PHE43 CAVITY OF HIV-1 ENV GP120, DISTINCT FROM THE FOSTEMSAVIR BINDING SITE. IN ADDITION, WE DISCOVERED THAT SOME OF THE MOST ACTIVE GP120 ANTAGONISTS ARE ALSO ACTIVE AGAINST HIV-1 REVERSE TRANSCRIPTASE (RT). HOWEVER, BECAUSE THE PHE43 CAVITY IS VERY NARROW AND CANNOT ACCOMMODATE ANY LARGER SCAFFOLDS, WE HYPOTHESIZE THAT MODIFICATIONS TO INCREASE THE RT-INHIBITORY ACTIVITY WOULD RESULT IN A LOSS OF GP120-TARGETED ENTRY INHIBITORY ACTIVITY AND VICE VERSA. BASED ON THESE CONSIDERATIONS, WE DECIDED TO FOCUS ON OPTIMIZING THE GP120-ANTAGONISTIC ACTIVITY IN THE CURRENT PROPOSAL. WE GAINED EXTENSIVE KNOWLEDGE OF THE 3D STRUCTURAL FEATURES FROM RESOLVING THE CRYSTAL STRUCTURES OF GP120–ANTAGONIST COMPLEXES. WE ALSO IDENTIFIED CRITICAL 2D STRUCTURAL FINGERPRINTS IN THE INHIBITORS THAT MADE THEM STRONG GP120 ANTAGONISTS WITH WEAK RT INHIBITORY ACTIVITY. WE HYPOTHESIZE THAT THESE NOVEL FINDINGS FROM STRUCTURAL ANALYSES WILL HELP TO DISSECT THE MECHANISM OF THESE INHIBITORS AND PAVE THE WAY TO DESIGN AND OPTIMIZE LEAD COMPOUNDS TO A NOVEL CLASS OF ENTRY INHIBITORS TARGETING SPECIFICALLY THE PHE43 CAVITY OF GP120. WE AIM TO DEVELOP 2–3 HIV-1 ENTRY INHIBITORS AS POTENTIAL PRECLINICAL AND CLINICAL CANDIDATES. THIS WELL-COORDINATED PROPOSAL IS EXPECTED TO GENERATE HIGHLY POTENT, CLINICALLY RELEVANT, ORALLY AVAILABLE DRUGS AS HIV-1 ENTRY INHIBITORS AND EFFECTIVE AGAINST RESISTANT MUTANTS. IN ADDITION, THESE NOVEL ENTRY INHIBITORS ARE EXPECTED TO ENRICH THE AVAILABILITY OF DRUGS THAT PREVENT VIRUS ENTRY BY TARGETING GP120 AND SERVE AS A NEW ARSENAL FOR COMBINATION THERAPIES, ESPECIALLY IN TREATMENT-EXPERIENCED PATIENTS, CONTRIBUTING TO THE FORMULATION OF LONG-ACTING DRUGS.

FUNCTIONAL INTERPLAY BETWEEN BRUGIA AND ITS WOLBACHIA SYMBIONT

AUTOPHAGIC REGULATION OF INFLAMMASOME-MEDIATED HYPERACTIVE STATE IN LIVING MACROPHAGES - ABSTRACT NUCLEOTIDE-BINDING DOMAIN (NBD) AND LEUCINE-RICH REPEAT (LRR)-CONTAINING PROTEINS (NLRS) ASSEMBLE INTO FUNCTIONAL SUPRAMOLECULAR ORGANIZING CENTERS CALLED THE INFLAMMASOMES, WHICH SERVE AS AN INTERFACE OF HOST DEFENSE AGAINST PATHOGENS OR OTHER TYPES OF DANGER. CANONICAL INFLAMMASOMES ACTIVATE CASPASE-1, AN INFLAMMATORY PROTEASE THAT UPON ACTIVATION, TRIGGERS TWO IMPORTANT BIOLOGICAL RESPONSES: 1) PROCESSING OF CYTOKINE INTERLEUKIN-1B (IL-1B), AND 2) FORMATION OF PLASMA MEMBRANE GASDERMIN D PORES TO POTENTIATE INFLAMMATORY CELL DEATH (PYROPTOSIS). CYTOKINE SECRETION AND PYROPTOSIS ARE COUPLED UNDER A STEADY-STATE CONDITION, AS THE GSDMD PORES ON THE PLASMA MEMBRANE, WHICH PERMEATES IL-1B, CAN ALSO COMPROMISE THE CELL MEMBRANE INTEGRITY LEADING TO THE LYTIC CELL DEATH. INTERESTINGLY, IN A HYPERACTIVE STATE FOLLOWING INFLAMMASOME ACTIVATION, CELLS SHOW SUSTAINED CYTOKINE SECRETION BUT RETAIN VIABILITY, EVEN THOUGH THIS PROCESS IS DEPENDENT ON GSDMD PORES FORMED ON THE PLASMA MEMBRANE. THE UNDERLYING MECHANISMS WHICH RECALIBRATE GSDMD TO ONLY SUSTAIN THE RELEASE OF THE INTERLEUKIN, BUT NOT PYROPTOSIS, ARE POORLY UNDERSTOOD. AUTOPHAGY IS REGARDED AS AN IMPORTANT REGULATOR OF INFLAMMATION. IN OUR RECENT STUDIES, WE FOUND THAT INFLAMMASOMES ARE REGULATED BY AN AGGRESOME-LIKE MECHANISM THAT PROMOTES INFLAMMASOME ASSEMBLY (YANG) BUT ALSO INDUCES AUTOPHAGY TO DAMPEN CASPASE-1 PROCESSING AND IL-B SECRETION (YIN) (MAGUPALLI ET AL., SCIENCE, 2020). OUR STUDIES ARE CONSISTENT WITH PREVIOUS REPORTS SHOWING THAT MICE LACKING THE KEY AUTOPHAGIC GENE, ATG16L1, IN HEMATOPOIETIC CELLS, WERE HIGHLY SUSCEPTIBLE TO DEXTRAN SULPHATE SODIUM-INDUCED ACUTE COLITIS, WITH THE SECRETION OF A HIGH AMOUNT OF IL-1B AND IL-18 CYTOKINES. IN ADDITION, THESE INFLAMMATORY PHENOTYPES WERE ALLEVIATED BY INJECTION OF ANTI-IL-1B AND IL-18 ANTIBODIES IN MICE. COLLECTIVELY, OUR AND OTHER PUBLISHED RESULTS DEMONSTRATE THAT AUTOPHAGY DIRECTLY REGULATES THE EXTENT TO WHICH INFLAMMASOMES ACTIVATE DOWNSTREAM SIGNALS. HOWEVER, IT IS UNCLEAR IF AUTOPHAGY CAN REGULATE THE SWITCH BETWEEN A HYPERACTIVE STATE AND A PYROPTOTIC STATE OF MACROPHAGES. IN THIS APPLICATION, WE WILL INVESTIGATE THE POTENTIAL LINK BETWEEN AUTOPHAGY AND MACROPHAGE HYPERACTIVATION USING INFLAMMASOME ASSAYS AND CELLULAR IMAGING.

UNDERSTAND AND IMPROVE IRON DISTRIBUTION AND ERYTHROPOIESIS IN BETA-THALASSEMIA

TREATMENT OF SICKLE CELL ACUTE PAIN EPISODES WITH INTRAVENOUS GAMMAGBLOBULIN

MALARIA IN BRAZIL: RBC VARIANTS & PARASITE INVASION

MOLECULAR MECHANISMS OF FILARIAL ENDOSYMBIOSIS

REDUCING SEXUAL RISK AMONG AFRICAN AMERICAN HETEROSEXUAL MEN

MECHANISMS OF ABNORMAL GROWTH REGULATION IN PROSTATIC ADENOCARCINOMA USING ABI1/HSSH3BP1 CONDITIONAL KNOCKOUT MOUSE MODEL

REDUCING SEXUAL RISK IN AFRICAN AMERICAN MEN WHO HAVE SEX WITH MEN: COOKING CLUB

ACTIVATING AUTOPHAGY IN FILARIAL WORMS TO IDENTIFY NOVEL MACROFILARICIDES

A SEQUENTIAL MIXED METHODS STUDY EVALUATING THE INFLUENCE OF VIOLENCE ON HIV CARE AND VIRAL SUPPRESSION AMONG YOUNG BLACK AND LATINX MSM

A MODEL NICHE SYSTEM TO EXPAND PLATELETS FOR TRANSFUSION

A NOVEL AND EFFECTIVE NANOBODY TO PREVENT AND TREAT ZIKA VIRUS INFECTION

IMPACT OF GEOGRAPHIC MOBILITY ON PREP AND HIV CARE OUTCOMES AMONG LATINO GAY, BISEXUAL AND OTHER MEN WHO HAVE SEX WITH MEN - ABSTRACT HIV CONTINUES TO BE A MAJOR PUBLIC HEALTH CONCERN FOR HISPANIC/LATINO GAY, BISEXUAL AND OTHER MEN WHO HAVE SEX WITH MEN (GBMSM) IN THE U.S. IMMIGRANT/MIGRANT LATINO GBMSM MAKE UP THE LARGEST PROPORTION OF FOREIGN-BORN SEXUAL MINORITY POPULATION AND ARE A KEY GROUP VULNERABLE TO HIV ACQUISITION AND TRANSMISSION, YET ARE RARELY THE FOCUS OF FUNDED RESEARCH. IMMIGRANT/MIGRANT LATINO GBMSM ARE MORE LIKELY TO RECEIVE DELAYED HIV DIAGNOSES AND TO BE UNINSURED/UNDERINSURED THAN U.S.-BORN LATINOS, IN ADDITION TO FACING IMMIGRATION- RELATED BARRIERS TO CARE. SPATIAL SEGREGATION AMONG IMMIGRANT/MIGRANT LATINO GBMSM IS LINKED TO DIMINISHED PROXIMITY TO SPANISH-LANGUAGE PRE-EXPOSURE PROPHYLAXIS (PREP) AND HIV SERVICE NAVIGATION SERVICES AND REDUCED ACCESS TO CULTURALLY COMPETENT PREP AND HIV CARE, CREATING AN INCREASINGLY INVISIBLE HIV BURDEN. RATES OF NEW HIV DIAGNOSES AND HIV VIRAL SUPPRESSION VARY DRAMATICALLY BETWEEN COUNTRIES OF ORIGIN FOR IMMIGRANT/MIGRANT LATINO GBMSM. GEOGRAPHIC MOBILITY HAS BEEN SHOWN TO ENHANCE HIV VULNERABILITY AND ADVERSELY IMPACT HIV CARE AND TREATMENT IN PEOPLE LIVING WITH HIV, YET THERE IS LIMITED DATA ON HOW GEOGRAPHIC MOBILITY IMPACTS PREP AND HIV CARE OUTCOMES AMONG LATINO GBMSM IN THE U.S. FURTHER, GEOGRAPHIC MOBILITY MAY LEAD TO POSITIVE OUTCOMES, AS PEOPLE MAY TRAVEL FOR BETTER ACCESS TO HIV PREVENTION AND CARE SERVICES, OR MOVE TOWARD BETTER SUPPORT NETWORKS OR AWAY FROM HIV AND PREP STIGMA PREVALENT IN THEIR COMMUNITIES. TO ADDRESS THE KNOWLEDGE GAP REGARDING THE IMPACT OF GEOGRAPHIC MOBILITY FOR LATINO GBMSM ON PREP AND HIV CARE OUTCOMES AND TO INFORM FUTURE INTERVENTIONS, THE SPECIFIC AIMS FOR THIS EXPLORATORY PROPOSAL ARE: AIM 1: TO CHARACTERIZE PATTERNS OF GEOGRAPHIC MOBILITY IN THE PAST 3 YEARS, INCLUDING DESTINATIONS, TEMPORALITY (INCLUDING DURATION, FREQUENCY, OR SEASONALITY), PURPOSE, AND LEVEL OF PLANNING OVER TRAVEL AMONG 40 LATINO GBMSM NOT LIVING WITH HIV (ON/NOT ON PREP) AND 40 LATINO GBMSM LIVING WITH HIV (ON/NOT ON ART) IN NYC USING QUALITATIVE IN-DEPTH INTERVIEWS AND MOBILITY MAPS. AIM 2: TO DETERMINE HOW PAST-YEAR GEOGRAPHIC MOBILITY IMPACTS PREP CARE OUTCOMES (PREP INITIATION/ADHERENCE, RETENTION AND PERSISTENCE IN PREP CARE) AND HIV CARE OUTCOMES (ART INITIATION/ADHERENCE, RETENTION IN CARE, AND VIRAL SUPPRESSION) LONGITUDINALLY OVER 12 MONTHS. WE WILL ENSURE A RANGE OF LATINO GBMSM EXPERIENCES ARE INCLUDED, BY PURPOSIVE SAMPLING BY: LIVING WITH AND WITHOUT HIV, REGION OF BIRTH (DOMINICAN REPUBLIC, PUERTO RICO, MEXICO/CENTRAL AMERICA, AND SOUTH AMERICA), AND RECENT VS. NON-RECENT IMMIGRANT/MIGRANT TO THE U.S. OUR APPROACH IS INNOVATIVE, AS IT EXAMINES THE IMPACT OF GEOGRAPHIC MOBILITY ON PREP AND HIV CARE OUTCOMES AMONG LATINO GBMSM WHO HAVE BEEN DISPROPORTIONATELY AFFECTED BY HIV AND HAVE BEEN UNDERSTUDIED, APPLIES MOBILITY MAPS IN A NOVEL WAY, AND HAS POTENTIAL TO INFORM FUTURE DESIGN OF INTERVENTIONS TO IMPROVE PREP AND HIV CARE OUTCOMES IN THIS POPULATION.

INFLUENCE OF SOCIAL NETWORKS ON HIV TESTING AND PREP CARE AMONG YOUNG LATIN AMERICAN GAY, BISEXUAL, AND OTHER MEN WHO HAVE SEX WITH MEN - HIV CONTINUES TO BE A MAJOR, “INVISIBLE” PUBLIC HEALTH CRISIS AMONG YOUNG LATIN AMERICAN GAY, BISEXUAL AND OTHER MEN WHO HAVE SEX WITH MEN (GBMSM) IN THE U.S. LATIN AMERICAN GBMSM ARE RARELY THE FOCUS OF FUNDED RESEARCH DESPITE THEIR SUSCEPTIBILITY TO HIV ACQUISITION. RELEVANT GBMSM REPRESENTED 70% OF NEW HIV DIAGNOSES AMONG LATIN AMERICAN GBMSM IN 2022; YOUNG PEOPLE (13-29 YEARS OLD) ACCOUNTED FOR 43%. LATIN AMERICAN GBMSM HAVE BETTER HEALTH PROFILES THAN OTHER POPULATIONS YET RECEIVE DELAYED HIV DIAGNOSES DESPITE HIGH LEVELS OF HIV TESTING AND SIMILAR SEXUAL BEHAVIORS. CONCERNINGLY, YOUNG LATIN AMERICAN MEN REPORT SIGNIFICANTLY LOWER HIV TESTING RATES, AND PREP UPTAKE REMAINS EXCEPTIONALLY LOW AMONG YOUNG LATIN AMERICAN GBMSM OVERALL. SOCIAL NETWORKS CAN SHAPE SUSCEPTIBILITY TO HIV ACQUISITION BY PROMOTING AND INHIBITING HIV PREVENTION BEHAVIORS (E.G., PREP USE), INCLUDING AMONG YOUNG LATIN AMERICAN GBMSM. HOWEVER, RESEARCH ON THE IMPACT OF SOCIAL NETWORKS ON HIV TESTING AND PREP CARE AMONG YOUNG LATIN AMERICAN GBMSM REMAINS LIMITED. FOR LATIN AMERICAN PEOPLE, THE CHARACTERISTICS AND STRUCTURES OF THEIR LOCAL AND LONG-DISTANCE NETWORKS CAN UNIQUELY SHAPE HOW SOCIAL CAPITAL (E.G., ACTUAL OR POTENTIAL RESOURCES), SOCIAL BONDING (E.G., SUPPORT, COHESION, AND PERCEPTIONS OF BELONGINGNESS), AND NETWORK STRESS (E.G., NEGATIVE SOCIAL INTERACTIONS) EACH WORK TO AFFECT THEIR HEALTH AND HEALTHCARE UTILIZATION, INCLUDING PREP CARE. THIS STUDY WILL FOCUS ON BOTH POSITIVE AND NEGATIVE NETWORK FACTORS, UNDERSTANDING THAT SOCIAL TIES CAN BE BOTH SUPPORTIVE AND HARMFUL, WITH THE OVERARCHING GOAL OF IDENTIFYING NETWORK-BASED POINTS OF INTERVENTION FOR DEVELOPING AND ENHANCING SOCIAL NETWORK-BASED INTERVENTIONS TO INCREASE HIV TESTING AND PREP UPTAKE, ADHERENCE, AND RETENTION IN CARE. TO ADDRESS THE KNOWLEDGE GAP REGARDING THE INFLUENCE OF SOCIAL NETWORKS FOR YOUNG (16-29 YEARS OLD) LATIN AMERICAN GBMSM ON HIV TESTING AND PREP CARE, AND TO INFORM FUTURE SOCIAL NETWORK-BASED HIV PREVENTION INTERVENTIONS FOR THIS POPULATION, THE SPECIFIC AIMS FOR THIS EXPLORATORY R21 PROPOSAL ARE: AIM 1: TO CHARACTERIZE AND MAP THE SOCIAL NETWORKS OF 60 YOUNG LATIN AMERICAN GBMSM IN NEW YORK CITY (NYC), NOT LIVING WITH HIV, THROUGH IN-DEPTH INTERVIEWS AND MAPPING OF FOUR SOCIAL NETWORK HEALTH AREAS (GENERAL/SEXUAL HEALTH, ALCOHOL/DRUG USE, HIV TESTING, AND PREP USE) USING CONCENTRIC CIRCLES METHODOLOGY. AIM 2: TO DETERMINE HOW SOCIAL NETWORK-LEVEL FACTORS INFLUENCE HIV TESTING (AT BASELINE) AND PREP CARE OUTCOMES (PREP INITIATION, ADHERENCE, AND RETENTION) AMONG 60 YOUNG LATIN AMERICAN GBMSM OVER 6 MONTHS, FOCUSING ON THE ROLE OF SOCIAL CAPITAL, SOCIAL BONDING, AND NETWORK STRESS. WE WILL ENSURE A RANGE OF YOUNG LATIN AMERICAN GBMSM EXPERIENCES ARE INCLUDED, BY PURPOSIVE SAMPLING BY ON AND NOT ON PREP.

A NOVEL NANOBODY WITH GOOD DRUGGABILITY TO PREVENT AND TREAT MERS-COV INFECTION

BABESIA: EXTRACELLULAR VESICLES AND THEIR ROLE IN INTERCELLULAR COMMUNICATION

ROLES OF N-GLYCANS ON NEUTROPHIL BETA2 INTEGRINS IN PROGRESSION OF ACUTE LUNG INJURY - PROJECT SUMMARY ACUTE LUNG INJURY (ALI) AND ITS SEVERE FORM, ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS), ARE THE COMMON CAUSE OF RESPIRATORY FAILURE. DESPITE ALL MEDICAL INNOVATIONS, TREATMENT OF ARDS REMAINS AN UNSOLVED PROBLEM. NEUTROPHILS ARE THE FIRST IMMUNE CELLS INFILTRATED INTO LUNGS DURING ALI, WHERE NEUTROPHILS RELEASE PROTEINASES, CYTOKINES, AND OXIDANTS TO KILL INVADING MICROBES, AS WELL AS TO INJURE LUNGS. CLINICAL DATA AND ANIMAL MODELS HAVE PROVED THAT NEUTROPHIL RECRUITMENT AND FUNCTION INFLUENCES THE PROGRESSION OF ALI. THUS, INSIGHTS INTO THE FINE-TUNED NEUTROPHIL RECRUITMENT AND FUNCTION MAY PROVIDE A NOVEL APPROACH FOR THE TREATMENT OF ALI. INTEGRIN B2 PLAYS AN IMPORTANT ROLE IN THE REGULATION OF NEUTROPHIL RECRUITMENT AND NEUTROPHIL FUNCTIONS. AT INFLAMED SITES, THE INTRACELLULAR SIGNALING UPREGULATES THE INTEGRIN LIGAND-BINDING AFFINITY, WHICH ALLOWS LIGANDS TO BIND. LIGAND BINDING TO INTEGRIN B2 IN THE HIGH-AFFINITY STATE ARRESTS NEUTROPHILS, A PREREQUISITE STEP FOR NEUTROPHIL RECRUITMENT, AS WELL AS CAUSES INTEGRIN CLUSTER FORMATION (I.E. INTEGRIN VALENCY) TO ENHANCE NEUTROPHIL ARREST. CURRENTLY, THE INTRACELLULAR SIGNALING IS THE ONLY APPROACH TO REGULATE THE INTEGRIN LIGAND-BINDING CAPABILITY. THE EXTRACELLULAR DOMAIN OF INTEGRIN B2 IS DECORATED WITH N-LINKED GLYCANS. YET, WHETHER AND HOW N- GLYCANS REGULATE THE INTEGRIN B2-LIGAND BINDING HAS NOT BEEN STUDIED. IN ADDITION, THE RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS (RAGE) ON ALVEOLAR EPITHELIAL CELL TYPE I (AT1) IS A MAJOR MEDIATOR FOR INFLAMMATORY RESPONSES IN LUNGS. INTEGRIN AMB2 ON NEUTROPHILS IS A LIGAND FOR RAGE, HOWEVER, THE ROLE OF THE INTERACTION OF INTEGRIN AMB2 WITH RAGE IN THE PROGRESSION OF INFLAMMATION IN ALI IS UNKNOWN. OUR PRELIMINARY STUDIES FOUND THAT REMOVING N-GLYCANS INCREASED INTEGRIN ALB2-MEDIATED NEUTROPHIL ADHESION AND LIGAND BINDING-INDUCED NEUTROPHIL CELLULAR RESPONSES. FURTHERMORE, REMOVING SIALIC ACIDS INCREASED AMB2 BINDING TO RAGE. THUS, WE HYPOTHESIZE THAT 1) N-GLYCANS ON THE EXTRACELLULAR DOMAIN PREVENT INTEGRIN ALB2 TO ADOPT THE HIGH-AFFINITY STATE AND/OR LIMIT THE INTEGRIN CLUSTER FORMATION; AND 2) REDUCED GLYCOSYLATION OF INTEGRIN AMB2 FACILITATES ITS BINDING TO RAGE THAT MAY INCREASE INFLAMMATORY RESPONSES IN LUNGS. THUS, WE PROPOSE TWO AIMS TO TEST THE HYPOTHESES: AIM 1: DETERMINE IF N-GLYCANS ON INTEGRIN ALB2 NEGATIVELY REGULATE ITS LIGAND-BINDING AFFINITY AND VALENCY; AND AIM 2: DETERMINE IF GLYCANS ON AMB2 NEGATIVELY REGULATE RAGE-MEDIATED INFLAMMATORY RESPONSES IN AT1 CELLS. INTEGRIN B2 CONTAINING MUTATIONS ON N-GLYCAN BEARING SITES, A NOVEL TOOL TO MEASURING INTEGRIN AFFINITY STATES, AT1 CELLS, AND MOUSE MODELS OF ALI WILL BE USED TO TEST OUR HYPOTHESES. OUR PROPOSED STUDY WILL DECIPHER THE ROLE OF N-GLYCANS ON NEUTROPHIL B2 INTEGRINS IN THE PATHOGENESIS OF ALI, WHICH MAY LEAD TO THE DEVELOPMENT OF A NEW THERAPY FOR ALI.

CONTINUOUS IN VITRO CULTURE OF BABESIA MICROTI

B. SUBTILIS SPORE-DELIVERED M2E-FP-BASED MUCOSAL UNIVERSAL INFLUENZA VACCINES

CRITICAL NEUTRALIZING DOMAIN-BASED VACCINES AGAINST NEW SARS-LIKE VIRUS HCOV-EMC

EFFECTS OF EXOGENOUS HORMONE TREATMENT ON VACCINE RESPONSES - ABSTRACT: SEX DIFFERENCES IN IMMUNITY ARE DYNAMIC THROUGHOUT THE LIFESPAN AND CONTRIBUTE TO HETEROGENEITY IN RISK OF INFECTIOUS DISEASES AND RESPONSE TO VACCINATION. SEXUAL DIMORPHISM IS, IN PART, DRIVEN BY SEX HORMONES AND HAS BEEN DEMONSTRATED IN BOTH INNATE AND ADAPTIVE IMMUNITY; TESTOSTERONE HAS AN IMMUNOSUPPRESSIVE EFFECT WHILE ESTROGEN IS IMMUNOENHANCING. THUS, FEMALES TEND TO MOUNT STRONGER IMMUNE RESPONSES, EXHIBIT LOWER INFECTION RATES FOR A VARIETY OF PATHOGENS, AND DEMONSTRATE ELEVATED RESPONSES TO VACCINATION. HOWEVER, THE MOLECULAR MECHANISMS DRIVING ENHANCED IMMUNITY IN FEMALES ARE NOT WELL UNDERSTOOD, AND THE DEGREE TO WHICH EXOGENOUS SEX HORMONES CONTRIBUTE TO IMMUNE RESPONSE IS UNKNOWN. TO DISSECT THE ROLE OF FEMALE SEX HORMONES IN ENHANCED IMMUNITY WE PROPOSE TO STUDY THE IMMUNE RESPONSES OF ADULT TRANSGENDER WOMEN (TW, INDIVIDUALS WHO IDENTIFY AS WOMEN BUT WERE ASSIGNED MALE AT BIRTH) UNDERGOING GENDER-AFFIRMING HORMONE THERAPY (GAHT) WITH ESTROGEN. THIS WILL PROVIDE A UNIQUE OPPORTUNITY TO UNDERSTAND THE IMPACT OF SEX HORMONES ON THE IMMUNE SYSTEM GENERALLY, AND MORE SPECIFICALLY, IN RESPONSE TO THE UPDATED SEASONAL COVID-19 VACCINES. NO STUDIES HAVE EXAMINED INTERACTIONS BETWEEN COVID-19 VACCINES AND EXOGENOUS SEX HORMONE REPLACEMENT THERAPIES (HRT). WE HYPOTHESIZE THAT TW UNDERGOING FEMINIZING GAHT WITH ESTROGEN WILL DEVELOP ENHANCED IMMUNE RESPONSES THAT ALIGN MORE WITH THEIR GENDER IDENTITY THAN THEIR BIOLOGICAL SEX. UNTANGLING THE RELATIONSHIP BETWEEN IMMUNE RESPONSE AND GENDER IN TW MAY FACILITATE AND IMPROVE EVIDENCE-BASED OUTCOMES FOR THOSE WHO RECEIVE THE SEASONAL COVID-19 VACCINES IN THIS POPULATION, AND IN FUTURE STUDIES ALSO IN OTHER POPULATIONS THAT RECEIVE EXOGENOUS SEX HORMONE TREATMENTS. WE WILL COMPARE THE HUMORAL AND CELLULAR RESPONSES IN ADULT TW (21-49 YEARS) BEFORE AND AFTER IMMUNIZATION WITH THE UPDATED SEASONAL COVID-19 VACCINE, WHEN OFFERED, WITH THOSE ELICITED IN CLINICALLY- AND AGE-MATCHED CISGENDER MEN AND WOMEN WITH SIMILAR COVID-19 VACCINE UPTAKE AND PRIOR INFECTIONS HISTORIES, AND WHO ARE PLANNING TO RECEIVE THE UPDATED VACCINE AND BE BLED PRIOR TO IMMUNIZATION (BASELINE) FOLLOWED BY THREE BLEEDS 7-, 28- AND 180-DAYS POST VACCINATION. TOGETHER, THE AIMS COMPRISE A CRITICAL FIRST STEP TOWARDS DETERMINING IF GAHT WITH ESTROGEN HAS IMMUNOENHANCING EFFECTS. CLEAR ASSOCIATION OF SEX HORMONE LEVELS WITH IMMUNE RESPONSES TO VACCINES WILL PROVIDE IMPORTANT INSIGHTS INTO THE IMPACT OF ENDOGENOUS AND EXOGENOUS SEX HORMONES ON IMMUNE HEALTH. WE POSIT THAT PROVIDING ACCURATE, UNBIASED, BIOLOGICALLY SOUND, EVIDENCE-BASED INFORMATION ABOUT VACCINE BENEFITS OR RISKS WILL SUPPORT PERSONAL DECISION-MAKING REGARDING WELLNESS AND DISEASE PREVENTION AND MAY INFORM HEALTHCARE STRATEGIES BASED ON SEX AND EXOGENOUS HORMONE USE, ULTIMATELY BENEFITING PUBLIC HEALTH FOR ALL AMERICANS.

TRANSFUSION-ASSOCIATED IMMUNE HEMOLYSIS

TESTING A COMMUNITY-LEVEL INTERVENTION FOR YOUNG AFRICAN AMERICAN MEN

INNOVATIVE 3-D IN VITRO CULTURING SYSTEMS FOR FILARIAL WORMS

INFORMAL SOCIAL CONTROL OF PARTNER VIOLENCE IN DRUG USERS

DEVELOPMENT OF A RADIATION COUNTERMEASURE WITH SEX-DEPENDENT RADIATION RESPONSE. - PROJECT SUMMARY/ABSTRACT THE RESEARCH GOAL IS TO UNDERSTAND THE MECHANISM UNDERLYING SEX-SPECIFIC EFFECTS IN THE REGULATION OF RADIATION RESPONSE AND TO DEVELOP A NOVEL RADIATION COUNTERMEASURE THAT TARGETS SUCH DIFFERENCES. RADIATION RESPONSES CAN VARY BETWEEN SEXES. WOMEN HAVE A HIGHER LONG-TERM RISK OF RADIATION-INDUCED CANCERS THAN MEN. HOWEVER, FEMALE MICE REVEAL GREATER RESISTANCE TO RADIATION AND EXHIBIT STRONGER SHORT-TERM PROTECTIVE RESPONSES. SUCH DIFFERENCES IN HUMANS AND RODENTS UNDERSCORE THE NECESSITY FOR FURTHER RESEARCH TO UNCOVER THE UNDERLYING MECHANISMS AND THEIR IMPLICATIONS FOR RADIATION BIOLOGY AND MEDICINE. RADIATION COUNTERMEASURES ARE DESIGNED TO REDUCE DAMAGES. DESPITE EXTENSIVE RESEARCH, ONLY A FEW HAVE RECEIVED FDA APPROVAL. CHALLENGES PERSIST IN ADDRESSING LONG-TERM RADIATION EFFECTS AND UNDERSTANDING SEX-RELATED RESPONSES. OUR PUBLISHED AND PRELIMINARY STUDIES SHOWED FEMALE LXN KNOCKOUT (LXN-/-) MICE HAD INCREASED HEMATOPOIETIC STEM/PROGENITOR CELL (HSPC) SURVIVAL AND REGENERATION IN PHYSIOLOGICAL CONDITION BY DOWNREGULATING THROMBOSPONDIN 1 (THBS1). LXN DELETION ENHANCES RADIATION-INDUCED SURVIVAL BY MITIGATING HEMATOPOIETIC-ACUTE RADIATION RESPONSE AND DELAYED EFFECTS BY UPREGULATING BCL-2. SURPRISINGLY, THE MALE LXN-/- MICE HAD NO HEMATOPOIETIC CHANGES IN PHYSIOLOGICAL CONDITION, WHICH IS MECHANISTICALLY DUE TO THE SEX-SPECIFIC SILENCE OF THBS1 GENE EXPRESSION. WE SCREENED A NOVEL LXN INHIBITOR THAT SHOWS RADIATION MITIGATION EFFECT IN VITRO AND IN VIVO IN FEMALE MICE. WE HYPOTHESIZE THAT PHARMACEUTICAL AND GENETIC INHIBITION OF LXN MITIGATES RADIATION INJURIES IN A SEX-DEPENDENT MANNER. TWO AIMS WILL BE PROPOSED. AIM 1 IS TO DETERMINE THE CELLULAR AND MOLECULAR MECHANISMS OF LXN DELETION IN RADIATION MITIGATION IN MALE MICE. AIM 2 IS TO DETERMINE WHETHER LXN INHIBITOR IS A COUNTERMEASURE WITH SEX-SPECIFIC RADIATION MITIGATION EFFECT. THE RESULTS WILL ADVANCE OUR UNDERSTANDING OF THE MECHANISMS UNDERLYING SEX-SPECIFIC REGULATION OF RADIATION RESPONSE AND PRODUCE SIGNIFICANT IMPLICATIONS FOR ACCURATE RADIATION RISK ASSESSMENTS AND TARGETED TREATMENTS IN VARIOUS RADIATION EXPOSURE SCENARIOS.

STRUCTURAL AND FUNCTIONAL ALTERATION OF HOST RBCS BY BABESIA - BABESIA SPP., THE ETIOLOGIC AGENTS OF BABESIOSIS IN ANIMALS AND HUMANS, ARE INTRAERYTHROCYTIC PROTOZOAN PARASITES TRANSMITTED TO HOSTS BY IXODES TICKS. LONG KNOWN TO CAUSE DISEASE IN DOMESTIC ANIMALS, B. MICROTI AND B. DIVERGENS HAVE EMERGED AS A GROWING PUBLIC HEALTH CONCERN FOR HUMANS, CAUSING FULMINANT DISEASE IN IMMUNO- COMPROMISED AND ASPLENIC POPULATIONS AND AN ALARMINGLY HIGH RATE OF HOSPITALIZATIONS AND SOMETIMES FATALITY IN THESE INDIVIDUALS. MOST CLINICAL COMPLICATIONS ARE DUE TO THE ABILITY OF THE PARASITES TO STRIKINGLY ALTER PROPERTIES OF HOST RBCS. I HYPOTHESIZE THAT STRUCTURAL, MORPHOLOGICAL, RHEOLOGICAL, AND FUNCTIONAL CHANGES OCCURRING IN INFECTED RBCS (IRBCS) ARE THE RESULT OF THE EXPORT OF SPECIFIC BABESIA PROTEINS, INCLUDING PROTEASES, WHICH INTERACT WITH THE CYTOPLASMIC, MEMBRANE SKELETAL AND MEMBRANE COMPONENTS OF THE RBCS. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO ELUCIDATE THE ALTERATIONS TO THE HOST RBCS AND UNCOVER THE MECHANISMS UNDERLYING THESE CHANGES BY IDENTIFYING AND CHARACTERIZING THE MOLECULAR PLAYERS-BOTH IMPACTED RBC CYTOSKELETAL PROTEINS AND THE KEY PARASITE MOLECULES RESPONSIBLE FOR THE CHANGES, TO BUILD A COMPREHENSIVE PICTURE OF HOST RBC REMODELING DURING INFECTION. AIM 1 WILL CONDUCT MORPHOLOGICAL, RHEOLOGICAL, AND BIOPHYSICAL ANALYSES TO DEFINE ALTERATIONS OF IRBCS USING IMAGE FLOW CYTOMETRY ALONG WITH MACHINE LEARNING ANALYSIS, EKTACYTOMETRY AND STIMULATED EMISSION DEPLETION MICROSCOPY. CHANGES IN 5 KEY RBC CYTOSKELETAL PROTEINS ON INFECTION-BAND 3, SPECTRIN, ANKYRIN, PROTEINS 4.1 AND 4.2, WILL BE MAPPED. SPECIFIC ANTIBODIES WILL BE USED IN IMMUNO-CHEMICAL METHODS TO TRACK CHANGES IN THESE CYTOSKELETAL ELEMENTS UP ON INFECTION AND MASS SPECTROMETRY PERFORMED TO IDENTIFY SPECIFIC PHOSPHORYLATION AND CLEAVAGE MODIFICATIONS. AIM 2 WILL IDENTIFY AND CHARACTERIZE 2 PARASITE CYSTEINE PROTEASES AND 2 ASPARTIC PROTEASES INCLUDING THEIR PROCESSING, SUB-CELLULAR LOCALIZATION, IDENTIFICATION OF EXPORT SEQUENCE SIGNATURES AND DEVELOP MOLECULAR TOOLS FOR DOWNSTREAM FUNCTIONAL ANALYSIS. THE SPECIFIC INTERACTIONS OF THESE PROTEASES WITH THE HOST RBC CYTOSKELETON PROTEINS WILL BE STUDIED USING INSIDE-OUT RBC VESICLE ASSAYS, IMMUNOPRECIPITATION, AND SURFACE PLASMON RESONANCE TO IDENTIFY SPECIFIC INTERACTIONS AND DELINEATE THE DOMAINS REQUIRED FOR MODIFICATION OF HOST CYTOSKELETAL PROTEINS. OVERALL, THESE AIMS WILL PROVIDE INSIGHTS INTO MECHANISMS OF RBC REMODELING BY THE PARASITE AND ITS PHYSIOLOGICAL RELEVANCE IN BABESIOSIS, OPENING DOORS FOR NOVEL AND TARGETED CHEMOTHERAPEUTICS, IN LINE WITH NIAID’S MISSION TO TACKLE EMERGING DISEASES RELEVANT TO PUBLIC HEALTH. A COMPREHENSIVE MENTORSHIP TEAM (DRS. LOBO, NARLA, HILLYER, YAZDANBAKHSH, MONTERO, ALLRED, MONETTI AND NORTH) HAS BEEN ASSEMBLED TO ENSURE THAT BOTH SCIENTIFIC AND CAREER DEVELOPMENT GOALS ARE MET. THE IMMEDIATE GOAL IS TO GAIN ADVANCED TRAINING IN A RELATIVELY UNDERSTUDIED PARASITE USING MULTIPRONGED EXPERIMENTAL APPROACHES, ALONG WITH ACQUIRING SOFT SKILLS NEEDED FOR SCIENTIFIC INDEPENDENCE. THIS K99/R00 GRANT WOULD EVENTUALLY PROVIDE DATA AND MOLECULAR TOOLS FOR R01-LEVEL FUNDING, FULFILLING LONG-TERM CAREER GOALS FOR LAUNCHING AN INDEPENDENT CAREER IN STUDYING HOST-PARASITE INTERACTION IN BABESIOSIS.

EVALUATING ACCESSORY CELLS TO IMPROVE HOMING AND ENGRAFTMENT EFFICIENCY OF CD34+ CELLS FROM SICKLE CELL DISEASE PATIENTS - AS A CURE FOR THE LIFE-THREATENING HEMOGLOBINOPATHY OF SICKLE CELL DISEASE (SCD), GENE THERAPY HAS IMMENSE POTENTIAL, AVOIDING THE BURDENS WITH ALLOGENEIC TRANSPLANT OF FINDING AN APPROPRIATE DONOR AND THE RISK OF GRAFT VERSUS HOST DISEASE. COLLECTING AN ADEQUATE NUMBER OF HEMATOPOIETIC STEM CELLS (HSCS) FOR GENE MODIFICATION, HOWEVER, OFTEN REQUIRES AT LEAST 2 DAYS AND SOMETIMES MULTIPLE CYCLES OF LEUKAPHERESIS, IMPOSING A COST AND LOGISTICAL BURDEN. CHEMOTHERAPY CONDITIONING PRIOR TO INFUSION OF THE MODIFIED HSCS IS MYELOABLATIVE WITH WEEKS UNTIL NEUTROPHIL, LYMPHOID, AND PLATELET RECOVERY, IMPOSING INFECTIOUS AND TRANSFUSION-RELATED RISKS. THESE ISSUES ARE SIGNIFICANT QUALITY OF LIFE CONCERNS IN THE UNITED STATES, WHERE ABOUT 100,000 PEOPLE LIVE WITH SCD, AND ARE LIKELY TO LIMIT ACCESS TO SCD GENE THERAPY TO HIGHLY EXPERIENCED TRANSPLANT CENTERS. IN LOWER AND MIDDLE INCOME COUNTRIES SUCH AS IN SUB-SAHARAN AFRICA, WHERE ABOUT 236,000 BABIES ARE BORN EACH YEAR WITH SCD, HOWEVER, THESE ISSUES MAY BE INSURMOUNTABLE BARRIERS TO SCD GENE THERAPY IMPLEMENTATION. THIS PROPOSAL AIMS TO ADDRESS THESE IMPEDIMENTS TO EQUITY OF ACCESS TO SCD GENE THERAPY BY EVALUATING A LIKELY-TO-BE COST-EFFECTIVE AND EASY METHOD TO INCREASE HSC HOMING AND ENGRAFTMENT. IN THE NON-SCD AUTOLOGOUS TRANSPLANT SETTING, THE FLOW-THROUGH FRACTION AFTER CD34+ HSC IMMUNOMAGNETIC SELECTION IS KNOWN TO CONTAIN ACCESSORY/IMMUNE CELLS ASSOCIATED WITH IMPROVED HSC HOMING AND ENGRAFTMENT. WE HYPOTHESIZE THAT, IN THE SCD AUTOLOGOUS TRANSPLANT SETTING, CO-INFUSING WITH THE CD34+ CELLS PART OF THE CD34-NEGATIVE FRACTION, WHICH IS NORMALLY DISCARDED AFTER IMMUNOMAGNETIC CD34+ CELL SELECTION, WILL ASSIST WITH HSC HOMING AND ENGRAFTMENT. USING THE IMMUNE AND ACCESSORY CELLS LEFT OVER FROM CD34+ CELL SELECTION OF SCD CORD BLOOD UNITS AND SCD PLERIXAFOR-MOBILIZED APHERESIS PRODUCTS, WE WILL IMMUNOPHENOTYPE THE ACCESSORY FRACTION AND DETERMINE IN VITRO HOMING EFFICIENCY OF A PRODUCT’S CD34+ CELLS INCUBATED ALONE (CONTROL ARM) AND WITH (EXPERIMENTAL ARM) ITS FLOW THROUGH CELLS (AIM 1). WE WILL ALSO PERFORM TRANSPLANT STUDIES IN IMMUNODEFICIENT MICE WITH CD34+ CELLS ALONE (CONTROL ARM) AND WITH (EXPERIMENTAL ARM) ITS FLOW THROUGH CELLS TO LOOK FOR IMPROVEMENT IN HUMAN CELL ENGRAFTMENT (AIM 2). ALTOGETHER, WE BELIEVE THAT EVALUATING THE EFFECTIVENESS OF ADDING BACK THE CD34- FLOW THROUGH AFTER CD34+ GENE MODIFICATION MAY OFFER A COST-EFFECTIVE METHOD FOR INCREASING EQUITY OF ACCESS TO SCD GENE THERAPY.

RATIONAL DESIGN OF M2E-FP CONSERVED EPITOPE-BASED UNIVERSAL INFLUENZA A VACCINES